BR102013033827A2 - multipotent and immunocompatible stem cell concentrate, use of concentrate, biopharmaceutical, dosage form, method of treatment and process of obtaining stem cell concentrate - Google Patents

Abstract

multipotent and immunocompatible stem cell concentrate, use of concentrate, biopharmaceutical, dosage form, method of treatment and process of obtaining stem cell concentrate. The present invention relates generally to a stem cell concentrate isolated from vascularized mammalian adipose tissue containing biopharmaceuticals for use in therapies applied to mammalian diseases.

Description

Multipotent and Immunocompatible Stem Cell Concentration, Use of the Concentrate, Biopharmaceutical, Dosage Form, Method of Treatment, and Process of Obtaining a Concentrate

The present invention relates generally to a stem cell concentrate isolated from mammalian vascularized adipose tissue, by the method described herein, biopharmaceuticals containing such a concentrate, and its use in therapies applied to diseases. of mammals.

In the context of this document the term "stem cells" is synonymous with multipotent stem cells, mesenchymal stem cells, medicinal signaling cells, stromal mesenchymal cells, and adult stem cells.

Precedents It is well known that stem cells have the ability to divide into culture for indefinite periods, and to originate specialized cells. They can give rise to many differentiated cell types, and can be used to treat many types of diseases. Adult stem cells in mammalian adipose tissue are also known. Such cells have already been isolated and cultured for use in cell therapies such as transplants.

However, to date, existing knowledge about such stem cells has not solved several drawbacks. Among them, the low productivity of the multiplication processes of such cells, in view of the needs of their use as well as little understanding as to the composition of stem cells used in cell therapies. In general, the fraction of stem cells isolated from adipose tissue is called the vascular stromal fraction of adipose tissue that derives from perivascular cells and also has parenchymal action properties similar to mesenchymal stem cells.

Also, the large amount of stem cells to be effective in various therapies, typically between 2x106 and 8x106 cells per kilogram of patient weight.

It may further be mentioned that even multipotent adipose-derived stem cells known in the prior art have been reported in limited cases of use in the treatment of certain types of diseases and tissues.

These facts make the use of adipose-derived stem cells in cell therapies limited and expensive, regardless of the fact that these cells are more abundant than those obtained from other sources. The present invention provides enhancements and solutions to such prior art situations as it provides: a high throughput process of immunocompatible multipotent stem cells derived from vascularized adipose tissue, including cell culture and division, - a synergistic concentrate of immunocompatible and multipotent cells which allows fewer stem cells to be used in a variety of therapeutic treatments for mammalian diseases (involving, for example, osteoarticular, muscular, neurological, hematological, ophthalmic, urinary system and many others).

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph of the increase in hematocrit levels following a single application of the cell concentrate of the invention; Figure 2 is a graph of the increase in leukocyte levels after a single application of the cell concentrate of the invention; Figure 3 is a graph of the increase in platelet levels after a single application of the cell concentrate of the invention; Figure 4 is a graph of the variation in animal glycemic level prior to treatment with the cell concentrate of the invention; Figure 5 is a graph of the variation in glycemic level of the animal after treatment with the cell concentrate of the invention; Figure 6 is a graph of the variation in the animal's glycemic level after the second transplant with the cell concentrate of the invention.

Description of the Invention The present invention, in a first aspect, relates to a multipotent and immunocompatible concentrate of medicinal molecule-secreting stem cells, derived from vascularized mammalian adipose tissue, comprising: immature stem cells (CTI), mesenchymal stem cells ( MSC) and perivascular cells (pericytes) The mammalian-isolated stem cell concentrate of the invention is immunopositive for at least CD146 +, cx-SMA +, CD44 +, CD73 +, CD90 +, CD105 +, CD13 +, vimentinaf, nestinal, Nanog + markers , Oct3 / 4 +, Sox2 + and immunonegative at least for the markers: CD34-, CD45-, CD56-, CD144-, CD14-, CD11b- and CD31-. Additionally, isolated from humans, the stem cell concentrate of the invention is immunopositive for NG2 +, PDGFR0 +, CD271 + (P75), CD29 +, SOX9 +, SOX17 +, F0X2 +, CD140 + and KLF4- negative markers.

Typically the stem cell contents in the concentrate of the invention are: - CTI: 1% to 20% - MSC: 70% to 100% - Pericytes: 10% to 30% Mention of "stem cell contents" refers to the amounts of cells expressing the respective markers (respectively from CTI, CTM or pericytes).

Particularly, without excluding any other, the concentrates of the invention have contents of 5% CTI, 100% CTM and 20% pericytes, or values close to them.

Immature stem cell (CTI), mesenchymal stem cell (CTM) and pericyte contents obtained from multiple adipose tissue stem cell niches vary among patients and vary with the isolation method.

Thus, mixtures of concentrates obtained from 2 or more donors may be used to achieve different mean levels from the individual ones. Alternatively, the stem cell concentrate may be enriched with specifically cultured pericytes. The cell composition in the concentrate of the invention is synergistic, since the combination of the effects of ITC, MSC and pericytes unexpectedly results in the use of fewer cells compared to the amounts of stem cells currently used for efficacy. in therapeutic treatments as described below.

Variations in the concentrate stem cell composition are also important because depending on the treatment of disease, trauma or injury, or aesthetic-dermatological appearance, this composition may be purified and / or enriched with cells expressing the important markers for each type of disease. illness, trauma, injury or other. A therapeutic composition can be adjusted to the patient's needs in relation to the proportion of markers expressed by the concentrate cells, as well as to the stem cell dosage, ie personalized medicine is proposed.

The concentrate cells of the invention may be used in their undifferentiated stem cell state, just as they may be induced to produce precursors of a specific lineage (e.g. glial cells) of interest for treatment, which may be purified. and applied as a pure population of precursors that are committed to cell differentiation of a single type, aiming at the benefit of the autocrine effect of these cells. On the other hand, these cells have paracrine effects that are produced by cells when they are not differentiated. For a broader therapeutic effect, the composition of undifferentiated cells may be applied together with the precursor cells.

These precursor cells have the ability to differentiate in vitro at least into derivatives of two embryonic leaflets: mesoderm (bone, cartilage, muscle, etc.) and ectoderm (neural cells). However, their differentiation is not limited to only mesoderm and ectoderm, because under appropriate conditions, these cells can also produce endoderm cell types, which characterize them as multipotent cells.

The cells contained in the concentrate of the invention have similar phenotypic and functional characteristics, but are distinct in many respects (cell division, expression of some genes and proteins, as well as paracrine action) from those present within the donor organism (in vivo).

Significant differences exist between the inventive concentrate and adipose tissue cell lines in vivo. Post-differentiation levels of TNFα and leptin secreted factors in (in vitro) culture of adipose tissue cells are much lower when compared to in vivo tissue. In addition, an important early regulator of adipogenesis transcription, KLF4, has significantly higher expression in vivo than in vitro.

Thus, it is not a matter of merely isolating stem cells as they are in the donor organism tissue, but rather removing them, treating them and multiplying them specifically so that they acquire the improved and appropriate characteristics. for the therapeutic purposes of the invention. The stem cell concentrate of the invention does not induce an immune response and can be used for autogenic (patient is donor and recipient), allogeneic (donor is related or same species as patient), and xenogeneic (stem cells from human different, unrelated).

Furthermore, the use of the cell concentrate of the invention in therapeutic treatments does not generate side effects, such as those typically observed in traditional therapeutic treatments using synthetic compounds, either short or long term. The stem cell concentrate of the invention is particularly suited to cell therapies, particularly cell transplants. The stem cell concentrate of the invention can derive from a broad spectrum of tissues derived from the three embryonic leaflets (mesoderm, ectoderm and endoderm).

A particular aspect of the invention relates to the standardization that must be pursued in its various aspects to ensure the success of the effects obtained, both in terms of the large production of cells from vascularized adipose tissue, as well as in the potency and therapeutic efficiency obtained with use of fewer cells compared to the state of the art.

Within an important aspect of the invention is the quality of the donor as a source of adipose tissue. In the veterinary area, the isolation of stem cells from young and healthy animals provides effective results regarding the high productivity, potency and therapeutic efficiency, compared to the isolation of cells from adult and / or elderly animals, or with already installed diseases, which provide unsatisfactory effects. Thus, for optimal implementation of the invention, the appropriate age of the donor mammal is between 20% and 30% of its total life. In particular, for dogs and cats donors of vascularized adipose tissue are suitable as optimal ranges of donor ages up to 2-3 years of life. For horses, up to 6-7 years of life.

Still in the veterinary area, a suitable place of choice for adipose tissue biopsy is lateral flank region or lateral surface of the posterior limb, but collection is not limited to these regions, as long as they allow the collection of vascularized adipose tissue. . Advantageously the collection of adipose tissue can be performed during elective surgical procedures in dogs and cats - for example, female castration (ovarian salpingo sterectomy) and pre-scrotal and inguinal castration of males. This avoids the need for a specific surgical procedure to take a sample, avoiding further pain and suffering of the patient. For the high throughput process of obtaining the stem cell concentrate of the invention it is sufficient to collect a small fragment of vascularized visceral fat, for example, between 0.1 and 0.3 cm3, as a 0.5x0.5x0 fragment, 5 cm. The small size of the collected sample also facilitates, where necessary, the transport of the sample to the laboratory (eg using a kit adapted for this purpose) and its preservation until the initiation of an in vitro cell isolation procedure. The stem cell concentrate of the invention may be cryopreserved, in a manner known to the person skilled in the art, to be stored for later use, in which it will be subjected to a thawing procedure also known to the person skilled in the art. For example, the stem cell concentrates of the invention may be frozen at the concentration of 1x106 or 2x106 in cryotubes and kept at -80 ° C for three months and then transferred to liquid nitrogen at -196 ° C. The culture medium used to cryopreserve cells keeps them viable and preserves their potential for differentiation, for example composed of 45% DMEM-h (Dulbecco's Modified Eagle Medium-high glucose), 45% fetal bovine serum and 10% dimethyl sulfoxide.

With regard to the application of the invention in the veterinary field, on the health aspect of the animal fat donor, it is advantageous to ensure optimal effects of the invention that animals (particularly dogs, cats and horses) are up to date with deworming to nematodes and cestodes, and with the following vaccinations: - dogs: distemper virus, hepatitis, parovirus, 4 strains of leptospirosis, coranivrose, parainfluenza, laryngotracheitis, giardia, kennel cough and anti-rabies; - cats: rhinotracheitis virus, calicivirosis, panleukopenia, feline leukemia, chlamydiosis, anti-rabies. - horses: rabies virus, equine influenza, encephalomyelitis.

Within another aspect, the invention relates to the use of the stem cell concentrate of the invention characterized in that it is in the preparation of products useful in the treatment of diseases, trauma, injuries or aesthetic-dermatological aspects with cell transplantation. Without excluding any other, examples of such products are biopharmaceuticals, solutions, tablets, ophthalmic formulations, formulations for topical and mucosal application, etc.

Within another aspect, the invention relates to biopharmaceuticals, characterized in that it comprises said stem cell concentrate, and one or more biologically acceptable ingredients, for example physiological solution, biomaterials (e.g. polymeric biomaterials), growth factors. (eg VEGF, TGF-beta, ITK), mononuclear cells, platelet-rich plasma, fibrin, hydroxyapatite, collagen membranes, bioactive molecules (eg hormones and mitogens).

Typically, an injection formulation minimally comprises the stem cell concentrate of the invention and physiological solution. The invention benefits from particular aspects of the process of obtaining the synergistic stem cell concentrate, for example, isolating a larger amount of stem cells from multiple adipotic cell niches in the same tissue fragment than in culture of explant releases in vitro unlimited amounts of stem cells in cultivation. Thus, fragments of adipose tissue initially plated in culture bottles can be transferred to new culture bottles in order to maintain the release of new stem cells.

Thus, without excluding any other, an advantageous process of obtaining synergistic stem cell composition of the invention comprises the following steps: A - obtaining a sample of vascularized mammalian adipose tissue, particularly of healthy young mammal; Lavagem washing and cleaning the tissue sample, for example with physiological solution and antibiotic; C - fragmentation of the tissue sample; D - gentle enzymatic digestion of adipose tissue fragments to eliminate adipocytes until inactivation of the enzyme, for example collagenase type I or IV; E - centrifugation to obtain cells of the vascular fraction of adipose tissue; F - tissue explant culture - particularly 5-7 tissue fragments per 25 cm 2 bottle of adherent material for 3 to 5 days without changing the culture medium; G - when reaching a confluence between 70 and 90%, dissociate via cellular enzymes the migrated cell colonies outside the tissue; H - optionally, the tissue separated in step E may be subjected to further explant cultivation, beginning a new step F; I - expansion of cells isolated in step G, particularly up to a maximum of 6 passages for later use in cell therapy.

After step A, the harvested tissue sample is prepared with cleaning and washing and may be placed in a container that serves as temporary storage medium for transport to another location for up to 48 hours. There are known kits in the state of the art for this purpose.

As already mentioned, to ensure optimal results of the invention, the vascularized adipose tissue donor mammal must be young and healthy. Advantageously, the donor's age should be up to a maximum of 30% of their total life, preferably up to 20%. Your health should be such that it essentially excludes any disease. The method employed allows to avoid any type of cell fluid filtration, or to dispense with any sort of cell selection using specific size, granularity or markers, for example magnetic beads and specific antibodies.

One aspect of enzymatic digestion of adipose tissue in step D is that vessels or fat from the sample are not discarded after digestion. The enzyme is suitable for the smooth digestion of connective tissue, for example type I or type IV collagenase, such as the GIBCO © product of Life Technologies, a US company.

If you wish to enrich the stem cell concentrate of the invention with pericytes, the blood vessels obtained from gentle digestion are separated from the adipose tissue sample, and explant culture is performed only with them according to steps F, G and I. Pericytes obtained are later added to the concentrate to be enriched.

In step F, it is preferred that the culture medium is not exchanged for 3-5 days, and an amount equivalent to the evaporated medium may be replaced. Maintaining the culture medium during this time provides selection only of cells that have the potential to adhere to the culture bottle plastic, a characteristic of mesenchymal stem cells. The confluence between 70 and 90% allows to isolate multiple spindle cell colonies with mesenchymal morphology of substantially standardized size.

To favor the good performance of the concentrate of the invention, the culture medium in step F must be of high quality, ie standardized, with a minimum of variation of its pre-identified components. A suitable example of such a culture medium, without excluding any other, is DMEM-h (Dulbecco's Modified Eagle Medium-high glucose), marketed under the name GIBCO © by the US company Life Technologies, supplemented with 15% fetal bovine serum from US company Hyclone, supplemented with 1% L-glutamine, 1% non-essential amino acids and 1% streptomycin / penicillin mixture to neutralize collagenase (all ingredients of US company Life Technologies). In step Η, the fragment of cultured adipose tissue can be transferred several times into the culture bottle, particularly up to 5 times, and continues to release cells.

It should be noted that in step I, cell expansion up to 6 passages is intended for safe use in cell therapy, preventing changes in their karyotype, proliferation or undifferentiated state from being induced. For other applications in basic and applied science the number of passages may be greater than the sixth pass.

In yet another aspect, the invention relates to the described stem cell concentrates for use in medical, veterinary or cosmetic therapy.

In another aspect, the invention relates to the use of the stem cell concentrate of the invention in therapies (autologous, heterologous and xenografting) for the treatment of mammalian diseases such as articular, neurological, haematological, ophthalmic, acute kidney disease. and chronic, musculoskeletal disease, diabetes, acute spinal cord injury.

Particularly in the veterinary field, therapies of pathologies such as: - hematopoietic diseases (aplasia and spinal cord hypoplasia), joint diseases (hip dysplasia, osteoarthritis, degenerative process) - fissures, failures and fractures - laceration may also be cited, without excluding any others. of tendon and ligaments - keratoconjunctivitis sicca, corneal ulcer - distemper virus-derived neurological sequelae - acute and chronic renal disease - masticatory myositis - type I diabetes - for dogs: distemper neurological sequelae and other neurodegenerative diseases, hip dysplasia, masticatory myositis ; - for dogs and cats: aplasia and spinal hypoplasia; bone fractures; degenerative osteoarticular diseases, acute spinal cord injury, acute and chronic kidney disease, eye diseases (eg retinal degeneration, kerate conjunctivitis sicca), diabetes; - for horses: tendon and ligament injuries, neurological sequelae promoted by encephalomyelitis virus, osteoarthritis and laminitis.

In another aspect, the present invention relates to dosage forms for the treatment of mammalian diseases, particularly the amount of stem cell concentrate cells ranging, for example, from 10x10 to 10x107 cells. trunk.

Tumorogenic Potential Evaluation The tumorigenic potential evaluation of the cell concentrate of the invention was made in a manner known to the person skilled in the art, for example from the information contained in Cell Cycle (2009). 15; 8 (16), 2608-2612 and "Teratoma formation: A tool for monitoring pluripotency in stem cell research" in www.stembook.org. The cell concentrate of the invention with 2x10 6 cells was injected into the pelvic limb of 5 nude mice. The mice were kept under normal conditions for a period of 60 days. After this period they were euthanized, and no newly formed mass was observed in the muscle and other organ.

Examples The following are examples of embodiments of the present invention. Such examples are to be construed solely as illustrative of particular embodiments of the invention, without limiting them in any way beyond those contained in the appended claims.

Example 1 - Isolation of inventive stem cells from dog and cat adipose tissue.

In the examples given below, obtaining a sample of vascularized adipose tissue followed the procedure below.

Firstly, the region of choice, lateral to the animal's hind limb, was well washed with soap and water. The skin was then scraped off using a razor or razor, cleaning the area again with soap and water. The area was then wiped with degermante chlorhexidine-moistened gauze (2% Riohex degermante chlorhexidine diglycerate solution, sold by the Brazilian company Rioquimica). This procedure was performed twice. Then 5 ml of lidocaine anesthetic were applied to the collection region. This region was first isolated with sterile field cloths and then incised into the skin with the aid of a 2 to 3 cm long scalpel. The fat fragment, about 0.5x0.5x0.5 cm, was removed using a scalpel and tweezers to ensure the presence of blood vessels in the sample. After collection, the site was cleaned with sterile gauze to dry possible bleedings and to suture the skin (thread), followed by disinfection after suturing, for example as rifocin. The collected adipose tissue was first washed in phosphate-saline buffer solution (PBS) with 5% streptomycin / penicillin to remove blood, debris and possible contaminants possibly present in the sample. The washing procedure was repeated 5 times. Then a sterile gauze was used to remove excess PBS contained in the sample. The tissue was transferred to a 60 mm diameter petri dish to fragment the tissue with the aid of a No. 22 scalpel blade to obtain several smaller sized fragments. Then, 2 ml of 0.075% type I or type IV collagenase (Gibco®) diluted in PBS at 37 ° C was added to the culture plate. The fragments together with collagenase were kept for 2 to 4 hours at 37 ° C under 5% CO 2 atmosphere. During the collagenase incubation, at least every 2 hours the sample was homogenized with the aid of a 1000 μΐ graduated pipette.

After this period, 5 ml of the following culture medium were added: DMEM supplemented with 15% fetal bovine serum from the US company Hyclone, supplemented with 1% L-glutamine, 1% non-essential amino acids and 1% streptomycin mixture. / penicillin to counteract collagenase (all ingredients of the US company Life Technologies). The contents were transferred to a 15 ml conical tube, and centrifuged for 5 minutes at 200g. This procedure was performed twice for complete inactivation of collagenase. In the last centrifugation the cells were resuspended in 1000 μΐ of the described culture medium and counted in a Neubauer chamber. Around 2x106 cells were transferred to a 25 cm2 culture bottle previously filled with 4,000 μΐ of the same culture medium.

Cells were maintained at 37 ° C in 5% CO2. After cell adhesion within 3 to 5 days the culture medium was discarded and new culture medium was added to the bottle. After a period of 3 to 5 days the first colonies of spindle-shaped fibroblast cells formed (counted as zero passage). In a period between 7 and 9 days the colonies reached between 70% and 90% of confluence. At this stage the first cell peal was performed in such a way that the contents of a 25 cm2 bottle were transferred to four 25 cm2 bottles - starting the first pass - which were then picked when reaching a confluence between 70 and 90%, being expanded ex vivo to passage 6 (from which a decline in cell proliferation rate was observed). Completion of these steps ensured the isolation of the cell concentrate of the invention.

In the following examples the cell concentrate of the invention used was obtained according to the above procedure, containing approximate contents of 5% CTI, 100% CTM and 20% pericytes, obtained by mixing concentrates from 3 different donors and enriched with pericytes. specifically cultivated (obtaining and isolating pericytes is done using a procedure known to the person skilled in the art).

In the equine examples, the adipose tissue sample was taken from the tail or withers of the animals.

Example 2 - Canine Distemper 2.1 Animals used The dogs used in this experiment were males and females of different breed, ranging in age from 1 to 6 years. All animals presented clinical neurological manifestations caused by the installation of distemper virus in the central nervous system, such as myoclonus, paraplegia, quadriplegia, paraparesis, seizure and inability to stay stationary and walk, as shown in table I below. All dogs used had previously undergone conventional treatment to combat symptoms manifested in the viremic phase (respiratory and digestive) and had no clinical manifestations of the gastrointestinal and respiratory system. Transplants were performed 30 days apart between each application. 2.2 Clinical evaluation before transplantation Patient history and complementary blood count, chest x-ray and abdominal ultrasound were performed to rule out the pre-existence of neoplasms. 2.3 Transplantation The inventive concentrate was transplanted intravenously into patients with distemper neurological sequelae. The number of cells varied with the weight of the animal (table I). Patients received on average three transplants of the inventive cell concentrate within 30 days of each other. For this purpose, intravenous access was performed with the aid of a catheter and the animal was kept under fluid therapy with 0.9% physiological solution. Previously cryopreserved cells were thawed according to the appropriate procedure and resuspended in 2 ml of 0.9% saline. Stem cell infusion was performed slowly.

Table I - neurological clinical manifestations of the patients (*) SRD: not defined race 2.4 Clinical evaluation after transplantation After treatment the animals were monitored for 1 hour by the veterinarian to observe possible anaphylactic reactions. No patient manifested rejection symptoms after transplantation of the inventive cell concentrate. Clinical returns were performed at 48 hours, 7 days and 21 days after CT application and the interval between one application and another was 30 days. Most animals had reduced neurological clinical signs and resumed walking - results were less positive in older animals with longer sequelae. Table II below shows the evolution of the reduction of neurological sequelae.

Table II Evolution of reduction of neurological sequelae of distemper after treatment with cells of the concentrate of the invention. DO- neurological symptoms before transplantation. Io transplant.

Dl- neurological symptoms after the 1st transplant (30 days) D2- neurological symptoms after the 2nd transplant (60 days) D3- neurological symptoms after the 3rd transplant (90 days) MT-thoracic member MP pelvic member Example 3 - Hypoplasia and aplasia bone marrow 3.1 Animals used Male dog of 30 kg, 1-year-old labrador, presented weakness, lack of appetite and on blood count found severe anemia. The patient had previously undergone monthly blood transfusions for a period of 5 months and was being treated with exogenous erythropoietin in an attempt to keep his hematocrit at acceptable levels for the canine species. 3.2 Clinical evaluation before transplantation Patient history and complementary blood count, chest x-ray and abdominal ultrasound were performed to rule out the pre-existence of neoplasms. Medullary biopsy diagnosed erythroid series granulocytic hypoplasia and megakaryocytic aplasia. 3.3 Transplantation A single 8x10 6 transplant of cells of the concentrate of the invention via intraosseous into the femur bone was performed. The animal was previously sedated with pre-anesthetic medication tramadol hydrochloride 4 mg / kg IM, followed by induction of propofol (2,6-diisopropylphenol) 8 mg / kg IV and maintenance with Isoflurane (2-chloro-2). - (difluoromethoxy) -1,1,1-trifluoroethane) to effect. Subsequently, wide lumbosacral trichotomy and antisepsis with 2% chlorhexidine were performed. After, the femoral crest was accessed with a needle for medullary biopsy. Cryopreserved cells were thawed, resuspended in 1 ml of 0.9% saline solution, and stem cell infusion was performed slowly. 3.4 Post-transplantation clinical evaluation Clinical returns were performed with peripheral blood collection for hematocrit control, in addition to the general physical examination, at intervals of approximately 30 days. The patient did not manifest rejection symptoms after transplantation of the cells of the invention. After a single transplant with the inventive cell concentrate the dog did not need to receive blood transfusion for one year - after that year bone marrow aspirate examination revealed a hematopoietically active bone marrow with slightly enlarged erythroid series with full maturation and ordered and predominance of mature forms, while the myeloid series was slightly diminished, with normal morphology, complete and ordered maturation and predominance of mature forms. The animal has remained well without transfusion ever since (until the filing of this patent application).

The graphs shown in figures 1, 2 and 3 show the significant increase in hematocrit (figure 1), leukocyte (figure 2) and platelet (figure 3) levels after a single application of the inventive cells.

Blood tests were performed at approximately 30-day intervals.

Example 4 -Diabetes Type I 4.1 Animal used Dog, 2-year-old female, 8 pounds, had high blood glucose levels, and was early diagnosed with type I diabetes. 4.2 Clinical Evaluation Pre-transplant Patient history and complementary blood count tests , chest x-ray and abdominal ultrasound to rule out the pre-existence of neoplasms. The glycemic test was performed daily, twice a day, confirming type I diabetes. To reduce glycemic levels, 3 to 6 units of insulin were administered daily to the patient daily. 4.3 Transplantation Four 4x10 6 cell transplants of the inventive concentrate were performed intravenously at 30-day intervals between each transplant. For this the cryopreserved cells were thawed and resuspended in 1 ml of 0.9% saline. Intravenous stem cell infusion was performed slowly. 4.4 Post-transplantation clinical evaluation After transplantation, the glycemic test was performed daily for twice a day for 60 days to monitor the reduction in glycemic level. Results demonstrated a reduction in glycemic levels and a reduction in insulin dose.

In figures 4, 5 and 6 the letter "T" means transplant date. Figure 4 is a graph of variation in animal glycemic level prior to treatment with stem cells. The gray color indicates the day the patient did not need to receive insulin, the dark gray color indicates the patient received insulin 3 units insulin a day, the white color indicates that the patient received 6 units insulin once a day. Figure 5 is a graph of the variation in animal glycemic level after treatment with stem cells. Note the reduction in the number of days without insulin administration and the dose. The light gray color indicates day when the patient did not need to receive insulin, the dark gray color indicates that the patient received insulin 3 units of insulin, the white color indicates that the patient received 6 units of insulin once a day. It is noted that the days without insulin increased, as well as the reduction in days that the dog needed to receive 6 units of insulin. Figure 6 is a graph of the variation in animal glycemic level after the second stem cell transplant.

Note the reduction in the large number of days without insulin administration and the dose. The gray color indicates the day the patient did not receive insulin, the white color indicates the patient received insulin 3 units of insulin.

It is noted that after the second transplant the dog reduced the daily insulin dose from 6 units per kilo to 3 units per kilo. In addition, the number of days without insulin was spaced. Example 5 - Masticatory Myositis 5.1 Animal used Dog, male, 11-year-old Labrador dog was unable to open its mouth, with swelling and difficulty feeding. 5.2 Clinical pre-transplant evaluation Patient history and complementary blood count, chest x-ray and abdominal ultrasound were performed to rule out the pre-existence of neoplasms. Chewing muscle biopsy revealed chewing muscle myositis. The opening capacity of the animal's jaw was 2.7mm. The dog had been treated with corticosteroids, but did not adapt to this treatment. 5.3 Transplant Three 8x10 6 stem cell transplants of the inventive concentrate of the invention were performed 30 days apart between each transplant. For this, cryopreserved cells were thawed, resuspended in 1 ml of 0.9% saline solution, and intravenous stem cell infusion was performed slowly. 5.4 Post-transplantation clinical evaluation After transplantation the animal was more willing, able to feed itself and the ability to use its mouth to pick up objects, an activity that was impossible to perform previously. The maxillary opening capacity has been expanded to 4.7mm.

Example 6 - Tendon Injury Of Horses Athletes 6.1 Animals used Three male horses ranging in age from 16 to 28 years had focal lesions on the superficial digital extensor tendon. The lesions were diagnosed with the aid of an ultrasound device and were classified according to the loss of the linear tendon fiber pattern as proposed by Nixon et al. , (Nixon AJ, Goodrich LR, Scimeca MS, Witte TH, Schnabel LV, Watts AE, et al. Gene therapy in musculoskeletal repair. Ann NY Acad Sci 2007; 1117: 310- 327) on a scale of 1 to 4. A Linear loss of tendon fiber pattern corresponding to 10 to 20% was classified as grade 1, while lesions between 25 to 50% were classified as grade 2.

In the table below are cited animals treated with the cell concentrate of the invention, their respective classifications according to the scale of the linear pattern of the tendon fiber and, according to the degree of tendon lesion cell number used in the transplant.

Table 1. Animals transplanted with the stem cell concentrate of the invention (according to example 1, horses being donors of adipose tissue), percentage of lesion and number of transplanted cells. 6.2 Transplantation The animals underwent trichotomy followed by povedine iodine antisepsis for subsequent transplantation of the concentrate cells of the invention. The route of application of the stem cells was at the site of the lesion, possible with the aid of ultrasound that allowed the exact identification of the lesion. Stem cell concentrate of the invention (horses were the donors of adipose tissue) was applied with the aid of a 40 x 12 needle and 3ml syringe. A single cell transplant was performed. 6.3 Clinical evaluation of the lesion after transplantation After 15 days, an evaluation of the stem cell transplantation of the inventive concentrate in the injured tendon area was performed with the aid of ultrasound.

A reduction in the extent of tendon tissue injury and collagen fiber organization was found. There was a significant improvement in the linear pattern of collagen fibers.

Example 7. Acute and chronic kidney disease 7.1 Animal used Dog, 13-year-old female pinscher with a history of poor appetite and low weight. Biochemical examination found elevated levels of urea 86 mg / dl and creatinine 2, 54 mg / dl. The patient was receiving fluid therapy twice daily and 2 Ml oral serum, with a special diet for renal patients with Royal renal (pate and ration) and white meat (chicken). The drug protocol used was: Ketosteril (amino acids and analogues, marketed by the Brazilian laboratory Fresenius), Glutamax (glutamine supplement, marketed by the Brazilian laboratory Vitafor), omeprazole, bromopride and Hemolitan (vitamin supplement marketed by the Brazilian laboratory Vetnil). 7.2 Clinical pre-transplant evaluation Patient history and complementary blood count, chest x-ray and abdominal ultrasound were performed to rule out the pre-existence of neoplasms. Biochemical examination and urinalysis were also performed, showing chronic kidney disease. 7.3 Transplant Two 2x10 6 cell transplants of the concentrate of the invention were administered intravenously 30 days apart. Cryopreserved cells were thawed and resuspended in 2 ml of 0.9% saline. THE

Cell infusion of the invention was performed slowly. The patient remained on conventional treatment for the disease during treatment with stem cells. 7.4 Post-transplantation clinical evaluation Clinical returns were performed at 48 hours, 7 days, and 21 days after transplantation with the stem cell concentrate of the invention and the interval between one application and another was 30 days. The patient did not manifest rejection symptoms after transplantation of the cells of the invention. Biochemical and urinalysis tests showed a reduction in blood levels of urea and creatinine, ionized calcium and an increase in hematocrit, as shown in the table below.

Table. Renal function values before and after transplantation. DO- laboratory data before the 1st transplant.

Dl- Laboratory data after 1st transplant (30 days) D2- Laboratory data after 2nd transplant (60 days) 8. Medullary Disc Extrusion Treatment 8.1 Animal used Dog, 9-year-old Doberman male with 39 kg absence of deep pain and paraplegia. 8.2 Clinical evaluation pretreatment Patient history and complementary blood count, chest x-ray and abdominal ultrasound were performed to rule out the pre-existence of neoplasms and through magnetic resonance imaging, the spinal cord extrusion in the thoraco-lumbar region was observed. T12-13 level TI3-1. 8.3 Transplantation After 21 days, the spinal cord was extruded into the thoracolumbar region. At this time (21 days) a single 4x10 6 stem cell transplantation of the inventive concentrate was performed. Cryopreserved cells were thawed and resuspended in 1 ml of 0.9% saline solution. Infusion of stem cells was performed slowly into the epidural space using a 3 ml syringe and a 20x5.5 needle. After stem cell therapy, we were given trama and dipyrone for 5 days and cephalexin for 14 days. On the 10th day after transplantation, the animal underwent physiotherapy for a period of 60 days three times a week and acupuncture once a week. 8.4 Post-transplantation clinical evaluation Clinical returns were performed at 48 hours, 7 days, and 21 days after transplantation with the stem cell concentrate of the invention. The patient showed no symptoms of rejection after transplantation. After 30 days of transplantation the animal kept the season and walked with difficulty and after 60 days the animal walked normally.

Example 9. Tendon laceration 9.1 Animal used Dog, 42-pound German Shepherd female, with traumatic injury to her left paw. The animal had difficulty walking because of its inability to support the left paw on the floor. 9.2 Clinical evaluation before transplantation Ultrasound revealed rupture of the common calcaneal tendon. 9.3 Transplantation After 28 days of traumatic tendon rupture, tenorrhaphy was performed and at the time of surgery the inventive concentrate was applied with 2x10 6 stem cells of the inventive concentrate. 9.4 Clinical evaluation after transplantation After 60 days ultrasound was performed, with complete organization of the tendon fibers, with the animal resuming motor activity. 10. Cerato Conjunctivitis Seca 10.1 Animal used Dog, a two-year-old male, of a defined breed, who had left eye tear production deficiency for 6 months. 10.2 Clinical assessment pretreatment Anamnesis of the patient was performed and the Schirmer test revealed a tear production level of 5mm. 10.3 Transplantation A single transplant of the stem cell concentrate of the invention was performed subconjunctivally into the main lacrimal gland and the lacrimal gland of the 3 eyelid. Cryopreserved cells were thawed and resuspended in 0.5 pL of 0.9% saline solution, and 0.3 pL was transfused into the main gland and 0.2 pL into the patient's third eyelid gland.

During treatment only the commercial tear was used within the first 7 days after initiation of stem cell transplantation. 10.4 Post-transplantation clinical evaluation Clinical returns were performed 7 and 14 and 21 days after transplantation with the cells of the invention. The patient showed no symptoms of rejection after transplantation of the stem cell concentrate of the invention. After 7 days the patient no longer used artificial tears and the schirmer test was 10mm and after 14 and 21 days 15mm, values considered at normal levels. 11. Ulcerative Keratitis 11.1 Animal used An eight-year-old female schnauzer dog had large bullous ulcerative keratitis in the right eye. 18.2 Clinical evaluation pretreatment Anamnesis of the patient was performed, and the inspection revealed large bullous ulceratitis in the right eye. Prior to treatment with stem cells, the patient was treated with antibiotics for 7 days. 11.3 Transplantation Five transplants of the stem cell concentrate of the invention were performed for 5 days, directly on the ulcerated cornea, of 1x10 6 cells resuspended in 5 µl saline. Cryopreserved cells were thawed prior to use. 18.2 Post-transplant clinical evaluation After one month of treatment with the stem cell concentrate of the invention, corneal transparency was maintained. When performing the visual acuity test (the threat test) the animal expressed a reaction and was thus considered positive for the test.

With the aid of the information and examples provided herein, the person skilled in the art can perform the invention in equivalent ways, i.e. not expressly described, but whose functions and results are of the same nature as those of the invention, therefore within the scope of the appended claims.

Claims (31)

1. MULTIPOTENT AND IMMUNOCOMPATIBLE TRUNK CELL CONCENTRATE, characterized in that it originates from vascularized mammalian adipose tissue, comprising immature stem cells, mesenchymal stem cells and pericytes. 2 . Concentrate according to claim 1, characterized in that it comprises contents of 1 to 20% of immature stem cells, 70 to 100% of mesenchymal stem cells, and 10 to 30% of pericytes. Concentrated according to Claim 1, isolated from mammals, characterized in that it is immunopositive for the markers CD146 +, α-SMA +, CD44 +, CD73 +, CD90 +, CD105 +, CD13 +, nestin +, Nanog +, Oct3 / 4. +, Sox2 + and immunonegative for the markers: CD34-, CD45-, CD56-, CD144-, CD14-, CD11b, CD31-. 4 Concentrated according to Claim 1, characterized in that, isolated from humans, it is immunopositive for the markers NG2 +, ββ, CD271 + (P75), CD29 +, SOX9 +, SOX17 +, FOX2 +, CD140 + and negative for the marker KLF4-. Concentrate according to Claim 1, characterized in that the mammalian donor of vascularized adipose tissue is young and healthy. Concentrate according to Claim 5, characterized in that the mammalian donor of vascularized adipose tissue is up to 20% to 30% of the maximum life span of such mammal. 7 Concentrated according to claim 5, characterized in that, when the donor is a dog or a cat, the age is up to 2 to 3 years. 8 Concentrated according to claim 5, characterized in that, when the donor is an equine, the age is up to 6 to 7 years. Concentrated according to claim 5, characterized in that the donor is substantially free of disease. Concentrated according to claim 1, characterized in that, for dogs or cats, the donor's vascularized adipose tissue is from the lateral side of the flank or the lateral surface of the hind limb and, for horses, from the withers or region. Tail Concentrate according to claim 1, characterized in that the vascularized adipose tissue of dogs or cats originates from pre-scrotal or inguinal castration of males, or ovary salpingo sterectomy, the castration of females. Concentrate according to Claim 11, characterized in that the vascularized adipose tissue of the donor mammal is a visceral fat fragment between 0.1 and 0.3 cm3. Concentrate according to any one of claims 1 to 12, characterized in that it is cryopreserved. Concentrate according to any one of claims 1 to 12, characterized in that it is derived from vascularized adipose tissue of two, three or more mammals, optionally enriched with pericytes. Concentrate according to any one of claims 1 to 14, characterized in that it is for use in medical, veterinary or cosmetic therapy. Use of the concentrate defined in any one of claims 1 to 15, characterized in that it is in the preparation of a product useful in transplanting cells for treating diseases. Use of the concentrate as defined in any one of claims 1 to 15, characterized in that said products are biopharmaceuticals, solutions, tablets, ophthalmic formulations, formulations for topical application. Use of the concentrate according to claim 17, characterized in that said product is for autologous, heterologous or xenograft therapy. Use of the concentrate according to claim 17, characterized in that said product is intended for systemic or local application such as intravenous, percutaneous, intraosseous, topical, topical and intrathecal. Use of the concentrate according to Claim 17, characterized in that the product is an eye drop. Use of the concentrate as defined in any one of claims 1 to 15, characterized in that it is in mammalian treatment therapies without neoplasia, which are not essentially suffering from any diseases other than the one to be treated, if any. 22 Use of the concentrate defined in any one of claims 1 to 15, characterized in that it is in mammalian treatment therapies among articular, neurological, hematological, ophthalmic, hematopoietic diseases, acute and chronic kidney disease, musculoskeletal disease, diabetes, acute spinal cord injury, tendon and ligament laceration, distemper virus-derived neurological sequelae, masticatory myositis, fissures, bone fractures and fractures, encephalomyelitis virus-promoted neurological sequel. BIOPharmacy comprising the stem cell concentrate of one or more claims 1 to 15 and one or more biologically acceptable ingredients. BIOPharmacy according to claim 23, characterized in that said ingredients are one or more of physiological solution, biomaterials, growth factors, mono-nuclear cells, platelet-rich plasma, fibrin, hydroxyapatite, collagen membranes, bioactive molecules. . Biopharmaceutical according to Claim 23, characterized in that said ingredient is a physiological solution. Dosage form for treating mammalian diseases, trauma, injuries, or aesthetic-dermatological features comprising the cell concentrate of any one of claims 1 to 15, with between 1 x 10 6 and 1 x 10 7 cells of said focused . Method of treating mammalian diseases, traumas, injuries or aesthetic-dermatological features by administering to a patient in need of treatment one or more amounts of the stem cell concentrate according to one of claims 1 to 15. TREATMENT METHOD according to claim 27, characterized in that said administration of said concentrate to treat bone marrow aplasia is by injection into the bone cavity. A treatment method according to claim 27, characterized in that said administration of said concentrate for treating ophthalmic diseases is by eye drop. Trunk cell concentrating process according to any one of claims 1 to 14, characterized in that it comprises the steps of: A - obtaining a sample of vascularized mammalian adipose tissue; B - washing and cleaning the tissue sample; C - fragmentation of the tissue sample; D - mild enzymatic digestion of tissue fragment fat; E - centrifugation to separate the tissue; F - tissue explant culture; G - when reaching a confluence between 70 and 90%, dissociate the colonies of migrated cells out of the tissue; H - optionally, the tissue separated in step E may be subjected to further explant cultivation, beginning a new step F; I - expansion of cells isolated in step G. Process according to Claim 30, characterized in that one or more of the following applies: in step A the donor mammal is young and healthy; in step A the adipose tissue sample is between 0.1 and 0.3 cm 3; in step B the washing and cleaning is done with physiological solution and antibiotic; in step D mild enzymatic digestion is done with type I or type IV collagenase; in step F the explant tissue culture is done with 5 to 7 tissue fragments per 25 cm 2 bottle of adherent material for 3 to 5 days without changing the culture medium; in step G the colonies are separated by enzymatic; in step Η, the cultured adipose tissue fragment may be transferred several times into the culture bottle; In step I, the expansion of the isolated cells is performed up to 6 passes.